Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. These vascular disturbances may increase morbidity and mortality in diabetic patients. Endothelial dysfunction from diabetes is associated with altered metabolism and inactivation of small (SKCa) and intermediate (IKCa) conductance calcium-activated-potassium channels in the animal and human coronary vasculature. However, the precise mechanisms responsible for diabetic inactivation of SKCa/IKCa and coronary endothelial dysfunction are still undefined. Recently, we demonstrated that elevation in intracellular NADH results in a significant decrease in endothelial SKCa/IKCa, and the lack of changes in SKCa/IKCa gene/protein abundances in the setting of diabetes and ischemia/reperfusion (I/R) suggests that the effect is post-translational. The goal of this project is to investigate how metabolic changes during diabetes negatively regulate SKCa/IKCa channels of animal/human endothelial cells and endothelial function in the animal/human coronary microvasculature and to evaluate if SKCa/IKCa activation and/or metabolic modulation protect endothelial cells/vessels against diabetes and ischemic insults. We hypothesize that persistent overproduction of reactive oxygen species (ROS) via NADPH oxidase (Nox), dysfunctional mitochondria and PKC during diabetes will result in 1) inactivation of endothelial SKCa/IKCa, 2) impairment of coronary endothelial function/arteriolar relaxation; and that 3) inhibition of Nox and mROS and/or PKC SKCa/IKCa overexpression may potentiate SKCa/IKCa activator-induced endothelial protection of endothelial cells/coronary arterioles against a simulated cardioplegia I/R injury. Using a type-2 diabetic mice model and heart/vessels/endothelial cell samples from patients, we will test our hypothesis by completing 4 specific aims. Aim 1 will investigate the molecular mechanisms by which persistent over-expression/activation of NADH/Nox during diabetes results in mROS and PKC overproduction/activation, leading to SKCa/IKCa inactivation, endothelial dysfunction/impaired vasodilatation, Aim 2 will elucidate the mechanisms by which persistent increases in mROS from the mitochondrial complex are required for diabetic inactivation of SKCa/IKCa, and endothelial function and arteriolar vasodilatation. Aim 3 will define the signaling pathways by which persistent PKC activation during diabetes negatively modifies SKCa/IKCa, and coronary endothelial function and microvascular relaxation. These experiments will also determine if PKC mediates its effects on the SKCa/IKCa channel either by direct action on the channel complex or by causing channel isolation from the sarcolemma. Aim 4: To examine if pharmacologic inhibition/gene knockdown of Nox, mROS, PKC and/or SKCa/IKCa overexpression may potentiate SKCa/IKCa activator-induced endothelial protection against a simulated cardioplegic I/R injury. To achieve these goals, multiple approaches will be employed such as patch clamping, molecular and cellular biology, biochemistry, vascular physiology, diabetic mouse model and human heart tissue/vessel/cell samples. The present study should lead to novel therapeutic strategies to preserve coronary endothelial function and microvascular relaxation for diabetic or non-diabetic patients with ischemic heart disease during cardiac surgery.